ATM kinase plays a central role in signaling DNA double-strand breaks

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM. 105628-07-7 were not activating ATM directly. The use of agents that disrupted chromatin structure without introducing breaks into DNA supported the hypothesis that gross alterations in chromatin structure were responsible for activation (Bakkenist and Kastan, 2003). The capacity of ATM to bind to DNA and associate with chromatin in response to radiation damage supports a role for ATM as a sensor of DNA damage (Andegeko that the Mre11 complex senses DNA DSBs and recruits ATM to broken DNA molecules. They showed that this complex was capable of activating ATM dimers to phosphorylate downstream cellular targets such as p53 and Chk2. Furthermore, depletion of Mre11 from extracts abrogated DNA DSBs and ATM-dependent phosphorylation of H2AX, suggesting that the Mre11 complex acts upstream of ATM activation (Constanzo (2006), who showed that ATM is activated by two independent steps both of which require the Mre11 complex. ATM activation has also been demonstrated to be dependent on phosphatase activity (Ali (2005) revealed that suppression of TIP60 histone acetyltransferase (HAT) blocks ATM activation and prevents ATM-dependent phosphorylation of p53 and Chk2. ATM forms a stable complex with TIP60 through a conserved FATC domain at its C-terminus. A second HAT, hMOF, also interacts with ATM and influences its function (Gupta phosphorylated peptide, from which S1981 was identified as the phosphorylation site, it was evident that other phosphopeptides were present in the ATM tryptic digest after exposure of cells to ionizing radiation (Bakkenist and Kastan, 2003). A separate study demonstrated the presence of 105628-07-7 at least seven phosphopeptides in ATM from irradiated cells using tryptic phosphopeptide mapping (Kozlov sites of phosphorylation, S367 and S1981. The S1981 site has been reported previously (Bakkenist and Kastan, 2003; Beausoleil 1593.8 by MALDI-TOF-MS. This signal was sensitive to treatment with Antarctic phosphatase (PPase). It disappeared, and … We initially determined whether the phosphorylation sites identified in ATM from irradiated cells could act as substrates for ATM kinase activity, Rabbit polyclonal to RB1 that is, autophosphorylation. The results in Figure 2A demonstrate that all three GST-fusion proteins containing the above three peptides are phosphorylated by ATM in response to radiation. This was expected for ATM1974C1992 105628-07-7 as it contains the pS1981 site (Bakkenist and Kastan, 2003) and for ATM363C375 as it contains the S367 site (ISQS), conforming to the general phosphorylation consensus sequence for ATM kinase (Kim and phosphorylation of Brca1 by ATM has been demonstrated at sites other than S/TQ (Cortez by 105628-07-7 demonstrating inhibition of GST-S1891ES1893E phosphorylation by wortmannin (Figure 2C). We provided evidence for ATM dependence in this reaction by showing that no radiation-induced phosphorylation occurred in an A-T cell line (Figure 2D). Figure 2 ATM phosphorylates S367, S1893 and S1981 in response to radiation-induced damage to DNA, we generated rabbit polyclonal antibodies that recognize this phosphorylation site (anti-pS1893). The specificity of this antibody was demonstrated by detection of a peptide containing pS1893 and no reactivity against the corresponding unphosphorylated peptide or a phosphorylated peptide containing pS1981 (Supplementary Figure 1A and B). This antibody did not detect pS1893 in cell extracts from unirradiated cells but a signal was clearly detectable in irradiated cells at 15 and 60 min post-irradiation (Supplementary Figure 1C). For comparison, we also employed an anti-pS1981 antibody, which detected an increase in phosphorylation in response to radiation (Supplementary Figure 1C) as described previously (Bakkenist and Kastan, 2003). Time-course analysis revealed that phospho-S1893 was detected.